WO2024077899A1 - 一种拟南芥叶片样本组织解离液及解离方法 - Google Patents

一种拟南芥叶片样本组织解离液及解离方法 Download PDF

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WO2024077899A1
WO2024077899A1 PCT/CN2023/087103 CN2023087103W WO2024077899A1 WO 2024077899 A1 WO2024077899 A1 WO 2024077899A1 CN 2023087103 W CN2023087103 W CN 2023087103W WO 2024077899 A1 WO2024077899 A1 WO 2024077899A1
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solution
dissociating
mannitol solution
arabidopsis leaf
arabidopsis
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王翠亭
张志明
肖云平
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上海欧易生物医学科技有限公司
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    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

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  • the invention belongs to the field of cell biology, relates to an Arabidopsis leaf sample dissociation method, and specifically relates to an Arabidopsis leaf sample tissue dissociation liquid and a dissociation method.
  • the tissues and organs of multicellular organisms are composed of a large number of highly specialized different cells, each of which has a specific phenotype and function. These special cell types are generated by undifferentiated progenitor cells through epigenetic reprogramming of the stem cell genome, establishing the unique transcriptional characteristics of each cell. Understanding the mechanism of cell differentiation is crucial to understanding the growth and development of multicellular organisms, the occurrence of diseases, etc., but our current understanding of the mechanisms of cell differentiation and development in the entire organism is still quite limited, largely because of the technical difficulty of purifying single cell types from tissues for transcriptional and epigenetic analysis.
  • the present invention provides a dissociation solution and a dissociation method for Arabidopsis leaf samples after extensive in-depth research.
  • the method provided by the present invention includes tissue surface cleaning, sample shearing, enzymatic digestion, mesh filtration, impurity removal and washing, and cell resuspension.
  • the present invention reduces the contamination of bacteria during the dissociation process by cleaning the leaves, and uses a self-developed tissue dissociation solution to improve the dissociation efficiency.
  • the invention provides an Arabidopsis leaf sample tissue dissociation solution.
  • the tissue dissociation solution is prepared by adding 1%-3% (W/V) cellulase and 1%-3% (W/V) pectinase into 8%-10% (W/V) mannitol solution.
  • W/V indicates mass volume concentration.
  • 8%-10% mannitol solution means that the mass volume concentration of mannitol in aqueous solution is 8%-10% g/mL
  • adding 1%-3% cellulase means that the mass volume concentration of added cellulase in 8%-10% mannitol solution is 1%-3%
  • adding 1%-3% pectinase means that the mass volume concentration of added pectinase in 8%-10% mannitol solution is 1%-3%.
  • the tissue dissociation solution is an 8% mannitol solution added with 2% cellulase and 2% pectinase.
  • the present invention also provides a method for dissociating Arabidopsis leaf samples, comprising:
  • step (2) Cut the sample into pieces: Place the leaves cleaned in step (1) in a sterile culture dish, add mannitol solution to submerge the leaves, and cut the leaves into thin strips;
  • tissue dissociation solution for enzymatic digestion to obtain protoplasts.
  • the tissue dissociation solution is an 8%-10% mannitol solution supplemented with 1%-3% cellulase and 1%-3% pectinase;
  • step (1) 2-3 leaves of 2-3 cm in length are taken from the Arabidopsis thaliana plant and the surface of the leaves is cleaned 2-3 times with a mannitol solution, each time for 2-3 minutes.
  • the enzymolysis temperature is room temperature and the enzymolysis time is 4-5 h; more preferably, the enzymolysis temperature is 25° C. and the enzymolysis time is 5 h.
  • step (3) the enzymatic hydrolysis is performed in a hybridization oven at a rotation speed of 10-15 rpm.
  • step (4) filtration is first performed using a 70 ⁇ m mesh and then using a 30 ⁇ m mesh.
  • the centrifugal force is 100 g
  • the centrifugal temperature is 4-10° C.
  • the centrifugal time is 2-5 min.
  • the specific step of resuspending is to discard the supernatant and keep 50 ⁇ l, add 1 ml of 8% mannitol solution, and mix by gently pipetting with a wide-mouthed rubber dropper.
  • the mass volume concentration of the mannitol solution used is 8%.
  • the present invention has the following beneficial effects:
  • the dissociation method of cleavage enzyme, cellulase and pectinase will produce a large number of ruptured protoplasts and a large number of chloroplasts, affecting the authenticity of the experiment, and the cell amount is 5 ⁇ 10 4 -1 ⁇ 10 5 , the protoplast yield is low and the chloroplast ratio is high, which is difficult to meet the subsequent experimental requirements.
  • the present invention can enzymatically hydrolyze the protoplasts within 5 hours, and the obtained protoplasts are greater than 1 ⁇ 10 6 , and the protoplast viability is more than 90%, and the chloroplast ratio is low. Therefore, the present invention has a significant improvement in the protoplast yield and the reduction of the chloroplast ratio.
  • Figure 1 shows Arabidopsis seedlings cultured under artificial greenhouse conditions for about one month.
  • Fig. 2 is a photo of the protoplasts obtained in Example 1 of the present invention.
  • Fig. 3 is a photo of the protoplasts obtained in Comparative Example 1 of the present invention.
  • FIG. 4 shows the sterile Arabidopsis tissue culture seedlings prepared in Example 2 of the present invention and Comparative Example 2 and cultured for 2 weeks.
  • Sample processing Six young leaves of about 2 cm in length were cut from Arabidopsis seedlings cultured for about one month under artificial greenhouse conditions (as shown in FIG1 ), and divided into experimental samples (3 leaves) and comparative samples (3 leaves).
  • the experimental sample (Example 1) was processed and protoplasts were dissociated using the method described in the present invention, and the comparative sample (Comparative Example 1) was processed and protoplasts were dissociated using conventional methods.
  • Example 1 The experimental method is as follows:
  • 8% mannitol solution 0.4 g mannitol (sigma, M9647-250G, the same product number is used below) is completely dissolved in 5 ml sterile water to obtain 8% mannitol solution (also used separately in the following steps);
  • step (3) Enzymatic digestion: Transfer the leaf strips obtained in step (2) into the prepared tissue dissociation solution, digest at 25°C for 4 hours, and obtain protoplasts by enzymatic digestion;
  • Cell resuspension Add 1 ml of 8% mannitol solution to the cell pellet after centrifugation in step (5) for resuspension. After microscopic examination, gently pipette with a wide-mouthed rubber-tipped pipette to mix. Adjust the protoplast suspension to 2 ⁇ 10 6 cells/ml by dilution or concentration.
  • Trypan blue staining was used for cell counting. 0.4% trypan blue was mixed with cell suspension at a ratio of 1:1. 10 ⁇ l was pipetted and added to the sample well of the hemocytometer for counting. The number of cells in the counting area was observed under a microscope. The calculation method was ((S1+S2+S3+S4+S5+S6+S7+S8+S9)/9/50*1000) cells/ ⁇ l. Note: S1, S2, S3, S4, S5, S6, S7, S8, S9 represent the number of cells in 9 counting areas respectively.
  • Example 1 using the dissociation method of the present invention, in Example 1, the protoplasts are in a single circular state of different sizes, the live protoplasts are transparent and clearly defined circles, and the dead protoplasts are stained blue under the light microscope. The cells are evenly dispersed in the visual field, without a large amount of agglomeration and cell debris, and the cell viability is good.
  • step (1) The leaf strips obtained in step (1) were transferred to a digestion solution containing 2% cellulase (sigma, C0615-1G), 1% pectinase (sigma, P2401-1KU), 0.4 M mannitol (sigma, M9647-250G), 20 mM MES (sigma, M3671-50G), 20 mM KCl (sigma, P9541-500G), 10 mM CaCl 2 (sigma, C4901-100G) and 0.1% BSA, and digested at 25°C for 4 hours to obtain protoplasts by enzymatic digestion;
  • Cell resuspension Add 1 ml of W5 solution to the cell pellet after centrifugation in step (4) for resuspension. After microscopic examination, gently pipette with a wide-mouthed rubber-tipped pipette to mix. Adjust the protoplast suspension to 2 ⁇ 10 6 cells/ml by dilution or concentration.
  • Trypan blue staining was used for cell counting. 0.4% trypan blue was mixed with cell suspension at a ratio of 1:1. 10 ⁇ l was pipetted and added to the sample well of the hemocytometer for counting. The number of cells in the counting area was observed under a microscope. The calculation method was ((S1+S2+S3+S4+S5+S6+S7+S8+S9)/9/50*1000) cells/ ⁇ l. Note: S1, S2, S3, S4, S5, S6, S7, S8, S9 represent the number of cells in 9 counting areas respectively.
  • the protoplasts were in a single circular state of different sizes.
  • the live protoplasts were transparent and clearly outlined circles, and the dead protoplasts were stained blue under the light microscope.
  • the cells were evenly dispersed in the field of view, but there were a large number of agglomerates and cell fragments, and the cell viability was low (as shown in Figure 3 and Table 1).
  • Example 2 and Comparative Example 2 Protoplast dissociation of sterile tissue culture seedling leaves of Arabidopsis thaliana
  • Example 2 Ten young leaves with a length of 1-2 cm were cut from sterile Arabidopsis tissue culture seedlings (as shown in Figure 4) that had been cultured for 2 weeks and divided into 2 groups (Example 2 and Comparative Example 2), with 5 leaves in each group.
  • Example 2 The experimental method is as follows:
  • the 8% mannitol solution and tissue dissociation solution used in this example are the same as those in Example 1.
  • step (4) Place the digested suspension in step (4) on crushed ice, then filter using a 70 ⁇ m (BD Falcon, 352350) mesh and then a 30 ⁇ m (Miltenyi, 130-098-458) mesh, centrifuge at 100G, 4°C, 5min, and discard the digestion solution;
  • Example 2 the protoplasts dissociated by the method of the present invention were in a single circular state of different sizes, the live protoplasts were transparent and clearly defined circles, and the dead protoplasts were stained blue under a light microscope. The cells were evenly dispersed in the field of view, without a large amount of agglomeration and cell debris, and the cell viability was good (as shown in Table 2).
  • step (1) The leaf strips obtained in step (1) were transferred to a digestion solution containing 2% cellulase (sigma, C0615-1G), 1% pectinase (sigma, P2401-1KU), 0.4 M mannitol (sigma, M9647-250G), 20 mM MES (sigma, M3671-50G), 20 mM KCl (sigma, P9541-500G), 10 mM CaCl 2 (sigma, C4901-100G) and 0.1% BSA, and digested at 25°C for 4 h to obtain protoplasts by enzymatic digestion;
  • Cell resuspension Add 1 ml of W5 solution to the cell pellet after centrifugation in step (4) for resuspension. After microscopic examination, gently pipette with a wide-mouthed rubber-tipped pipette to mix. Adjust the protoplast suspension to 2 ⁇ 10 6 cells/ml by dilution or concentration.
  • Comparative Example 2 the protoplasts prepared by the conventional dissociation method were in a single circular state of different sizes, the live protoplasts were transparent and clearly outlined circles, and the dead protoplasts were stained blue under the optical microscope. The cells were evenly dispersed in the field of view, but there were more agglomerates and cell fragments, and the cell viability was low (as shown in Table 2).

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Abstract

提供了一种拟南芥叶片样本组织解离液及解离方法。该组织解离液是在8%-10% g/mL甘露醇溶液中添加1％-3％ g/mL纤维素酶、1％-3％ g/mL果胶酶。使用该组织解离液解离拟南芥叶片样本时，能够提高拟南芥叶片的原生质体得率和减少叶绿体的占比。

Description

一种拟南芥叶片样本组织解离液及解离方法 技术领域
本发明属于细胞生物学领域，涉及拟南芥叶片样本解离方法，具体涉及一种拟南芥叶片样本组织解离液及解离方法。
 背景技术
多细胞生物的组织和器官由大量高度特化的不同细胞构成，每一种细胞类型都有特定的表型和功能。这些特殊的细胞类型是由未分化的祖细胞通过干细胞基因组的表观遗传重编程产生，建立起每个细胞独特的转录特征。了解细胞分化的机制对于理解多细胞生物的生长发育、疾病发生等至关重要，但目前我们对整个生物体中细胞分化、发育的机制了解仍然相当有限，很大程度上是因为从组织中纯化单个细胞类型以进行转录和表观分析的技术难度较高。
以往植物转录组学研究通常是将植物整个器官或组织均质化后测序，因此每个细胞对转录丰度的贡献变得不可识别。这种方法虽然有助于在器官或组织水平上解决许多生物学问题，但是无法了解发生在罕见细胞类型或单个细胞中的转录过程。采用单细胞测序技术，是研究植物单细胞水平转录信息的有效技术手段。不过由于植物细胞周围的细胞壁聚合物和细胞大小的巨大差异，在植物中进行单细胞转录组测序明显比在动物中更具挑战性。植物组织样本较小，解离出1×10 6数量的单细胞相对困难。文献报道的酶解方式多为纤维素酶过夜解离，该方法耗时长且对原生质体的损伤大，易产生大量的碎片，且过长的酶解时间导致细胞内的转录水平发生变化，不能反映原始样本中细胞真实的转录情况。因此解离出高质量的单细胞悬液尚有较大难度。
发明内容
为了克服现有技术存在的问题，本发明在进行了大量的深入研究后，提供了一种拟南芥叶片样本组织解离液及解离方法。本发明提供的方法包括组织表面清洗、样本剪碎、酶解消化、筛网过滤、去杂洗涤、细胞重悬，本发明通过对叶片进行清洗降低解离过程中杂菌的污染，采用自主研发的组织解离液提高解离效率。
为了实现本发明的目的，本发明采用的技术方案为：
本发明提供了一种拟南芥叶片样本组织解离液，所述组织解离液是在8%-10%（W/V）甘露醇溶液中添加1％-3％（W/V）纤维素酶、1％-3％（W/V）果胶酶。
其中，W/V（g/mL）表示质量体积浓度。“8%-10%甘露醇溶液”是指甘露醇在水溶液中的质量体积浓度为8%-10% g/mL，“添加1％-3％纤维素酶”是指添加的纤维素酶在8%-10%甘露醇溶液中的质量体积浓度为1％-3％，同样“添加1％-3％果胶酶”是指添加的果胶酶在8%-10%甘露醇溶液中的质量体积浓度为1％-3％。
优选地，所述组织解离液是添加有2％纤维素酶、2％果胶酶的8%甘露醇溶液。 
本发明还提供了一种拟南芥叶片样本的解离方法，包括：
(1)表面清洗：将拟南芥叶片样本用甘露醇溶液进行表面清洗； 
(2)样本剪碎：将步骤(1)清洗后的叶片置于无菌培养皿中，加入甘露醇溶液浸没过叶片、将叶片切成细条状； 
(3)酶解消化：将步骤(2)得到的叶片细条转移至组织解离液中酶解得到原生质体，所述组织解离液是添加有1％-3％纤维素酶、1％-3％果胶酶的8%-10%甘露醇溶液； 
(4)筛网过滤：将步骤(3)消化后的悬液置于碎冰上，然后筛网过滤、离心富集、弃消化液； 
(5)去杂洗涤：向步骤(4)得到的原生质体沉淀中加入甘露醇溶液离心洗涤； 
(6)细胞重悬：向步骤(5)离心后的细胞沉淀中加入甘露醇溶液进行重悬。
优选地，步骤(1)中，从拟南芥植株中取2-3cm长的叶片2-3片，用甘露醇溶液进行表面清洗2-3次，每次2-3min。
优选地，步骤(3)中，酶解温度为室温，酶解时间为4-5h；更优选地，酶解温度为25℃，酶解时间为5h。
更优选地，步骤(3)中，酶解在转速10-15rpm的杂交炉中进行。
优选地，步骤(4)中，先使用70μm筛网过滤，再使用30μm筛网过滤。
优选地，步骤(4)和(5)中，离心力100g，离心温度4-10℃，离心时间2-5min。
优选地，步骤(6)中，所述重悬的具体步骤为弃上清后留50μl，加入1ml 8%甘露醇溶液，用宽口的胶头滴管轻柔吹打混匀。
优选地，步骤(1) 、(2)、(5)和(6)中，使用的甘露醇溶液的质量体积浓度均为8%。
与现有技术相比，本发明的有益效果如下：
与现有离析酶与纤维素酶，果胶酶解离方法相比，离析酶与纤维素酶，果胶酶解离方法会产生大量破裂的原生质体和大量的叶绿体，影响实验的真实性，且细胞量在 5×10 4-1×10 5个，原生质体得率偏低且叶绿体占比高，难以满足后续的实验要求。本发明能够将原生质体在5h之内酶解下来，并且得到的原生质体大于1×10 6个，且原生质体活率在90％以上，叶绿体占比较低。因此，本发明在原生质体得率上与减少叶绿体的占比上有了明显的提高。
 
 附图说明
图1为人工温室条件下培养1个月左右的拟南芥幼苗。
图2为本发明实施例1中得到的原生质体照片。
图3为本发明对比例1中得到的原生质体照片。
图4为本发明实施例2和对比例2中准备的培养2周的无菌拟南芥组培苗。
 实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干或者多个变形和改进。这些都属于本发明的保护范围。
在一个具体的实施方式中，本发明所述方法的具体步骤为：
样本处理：从人工温室条件下培养1个月左右的拟南芥幼苗(如图1)，剪取6片约2cm长的幼嫩叶片，分为实验样本（3片）和对比样本（3片）。实验样本（实施例1）用本发明所述方法进行处理和原生质体解离，对比样本（对比例1）采用常规方法进行处理和原生质体解离。
实施例1实验方法如下：
组织解离液配制：
8%甘露醇溶液配制：0.4g 甘露醇(sigma，M9647-250G，以下均采用相同货号产品)完全溶解于5 ml 无菌水中，即得8%甘露醇溶液（以下步骤中亦单独使用）；
再向8%甘露醇溶液中依次加入0.1g(sigma，C0615-1G) 纤维素酶和0.1g 果胶酶(sigma，P2401-1KU)，充分溶解，即得组织解离液，备用。
实验步骤：
(1)表面清洗：从拟南芥植株中取2-3cm长的叶片2-3片，将叶片使用流动的25℃的8%甘露醇溶液冲洗表面2-3次，每次2-3min； 
(2)样本剪碎：在无菌培养皿中，加入8%甘露醇溶液使没过叶片（约5ml），用锋利的刀片小心将叶片充分切成细条状； 
(3)酶解消化：将步骤(2)得到的叶片细条转移至配制好的组织解离液中，25℃消化4小时，酶解得到原生质体； 
(4)筛网过滤：将步骤(3)消化后的悬液置于碎冰上，然后先使用70μm(BD Falcon，352350) 筛网，再使用30μm(Miltenyi，130-098-458) 筛网过滤，100G，4℃，5min离心，弃消化液；
(5)去杂洗涤：向步骤(4)得到的原生质体沉淀中加入1ml 25℃ 8%甘露醇溶液，100G，4℃，5min离心，弃上清，得到的原生质体沉淀中加入1ml 25℃ 8%甘露醇溶液，100G，4℃，5min离心，弃上清，留50μl； 
(6) 细胞重悬：向步骤(5)离心后的细胞沉淀中加入1ml 8%甘露醇溶液进行重悬，镜检后用宽口的胶头滴管轻柔吹打混匀，通过稀释或浓缩将原生质体悬液调整至2×10 6 cells/ml。 
结果及分析：
细胞计数采用台盼蓝染色，0.4％的台盼蓝与细胞悬液按1:1混匀，用移液枪吸取 10μl加到血球计数板的加样孔中计数，显微镜观察计数区的细胞个数， 计算方式为((S1+S2 +S3+S4+S5+S6+S7+S8+S9)/9/50*1000)个cells/μl。注：S1，S2，S3，S4，S5，S6，S7，S8，S9分别代表9个计数区的细胞个数。
如图2所示，采用本发明所述解离方法，实施例1中，原生质体呈单个不同大小的圆形状态，活原生质体为透亮且轮廓清晰的圆，光镜下死原生质体被染上蓝色。视野中细胞均匀分散开，无大量结团及细胞碎片，细胞活率良好。
对比例1实验方法如下：
(1) 样本剪碎：干净的培养皿中垫一张用无菌水湿润的滤纸，用锋利的刀片小心地将新鲜采集的3片拟南芥叶片充分切成细条状； 
(2)酶解消化：将步骤(1)得到的叶片细条转移至含2%纤维素酶(sigma，C0615-1G)、1%果胶酶 (sigma，P2401-1KU)、0.4M甘露醇(sigma，M9647-250G)、20mM MES（sigma，M3671-50G）、20mM KCl（sigma，P9541-500G）、10mM CaCl 2（sigma，C4901-100G）和0.1% BSA的消化液中，25℃消化4小时，酶解得到原生质体； 
(3)筛网过滤：将步骤(2)消化后的悬液置于碎冰上，然后先使用70μm(BD Falcon，352350)筛网，再使用30μm(Miltenyi，130-098-458) 筛网过滤，100G，4℃，5min离心，弃消化液；
(4)去杂洗涤：向步骤(3)得到的原生质体沉淀中加入1ml 4℃ W5溶液（154mM NaCl，125mM CaCl 2，5mM KCl，4mM MES，pH5.7），100G，4℃，5min离心，弃上清，得到的原生质体沉淀中加入1ml 4℃ W5溶液，100G，4℃，5min离心，弃上清，留50μl； 
(5)细胞重悬：向步骤(4)离心后的细胞沉淀中加入1ml W5溶液进行重悬，镜检后用宽口的胶头滴管轻柔吹打混匀，通过稀释或浓缩将原生质体悬液调整至2×10 6 cells/ml。 
结果及分析：
细胞计数采用台盼蓝染色，0.4％的台盼蓝与细胞悬液按1:1混匀，用移液枪吸取 10μl加到血球计数板的加样孔中计数，显微镜观察计数区的细胞个数， 计算方式为((S1+S2 +S3+S4+S5+S6+S7+S8+S9)/9/50*1000)个cells/μl。注：S1，S2，S3，S4，S5，S6，S7，S8，S9分别代表9个计数区的细胞个数。
如图3所示，原生质体呈单个不同大小的圆形状态，活原生质体为透亮且轮廓清晰的圆，光镜下死原生质体被染上蓝色。视野中细胞均匀分散开，但有大量结团及细胞碎片，细胞活率较低（如图3、表1所示）。
                                       表1实施例1和对比例1实验效果对比
实施例2及对比例2： 拟南芥无菌组培苗叶片原生质体解离
从培养2周的无菌拟南芥组培苗（如图4）上剪取1-2cm长的幼嫩叶片10片，分为2组（实施例2 和 对比例2），每组各5片叶片。
实施例2实验方法如下：
本实施例采用的8%甘露醇溶液及组织解离液均与实施例1相同。
实验步骤：
(1)加5ml室温的8%甘露醇溶液，冲洗掉无菌组培苗上剪取的5片叶片上沾染的培养基。
(2) 重复清洗一次，将叶片上沾染的培养基洗涤干净。
(3) 弃清洗液，加入8%甘露醇溶液5ml、用锋利的刀片小心将叶片充分切成细条状。 
(4) 将切好的叶片细条转移至组织解离液中，25℃消化4小时，酶解得到原生质体； 
(5) 将步骤(4)消化后的悬液置于碎冰上，然后先使用70μm（BD Falcon，352350）筛网，再使用30μm（Miltenyi，130-098-458）筛网过滤，100G，4℃，5min离心，弃消化液； 
(6) 向原生质体沉淀中加入1ml 25℃ 8%甘露醇溶液，100G，4℃，5min离心，弃上清，留50μl； 
(7) 加入1ml 8%甘露醇溶液进行重悬，镜检后用宽口的胶头滴管轻柔吹打混匀,通过稀释或浓缩将原生质体悬液调整至2×10 6 cells/ml。
结果及分析：
细胞计数采用台盼蓝染色，0.4％的台盼蓝与细胞悬液按1:1混匀，用移液枪吸取 10μl加到血球计数板的加样孔中计数，显微镜观察计数区的细胞个数， 计算方式为((S1+S2+S3+S4+S5+S6+S7+S8+S9)/9/50*1000)个cells/μl。注：S1，S2，S3，S4，S5，S6，S7，S8，S9分别代表9个计数区的细胞个数。
在实施例2中，采用本发明所述方法解离的原生质体呈单个不同大小的圆形状态，活原生质体为透亮且轮廓清晰的圆，光镜下死原生质体被染上蓝色。视野中细胞均匀分散开，无大量结团及细胞碎片，细胞活率良好（如表2所示）。
对比例2实验方法如下：
(1)样本剪碎：干净的培养皿中垫一张用无菌水湿润的滤纸，用锋利的刀片小心地将新鲜采集的5片拟南芥叶片充分切成细条状； 
(2) 酶解消化：将步骤(1)得到的叶片细条转移至含2%纤维素酶(sigma，C0615-1G)、1%果胶酶(sigma，P2401-1KU)、0.4M甘露醇(sigma，M9647-250G)、20mM MES（sigma，M3671-50G）、20mM KCl（sigma，P9541-500G）、10mM CaCl 2（sigma，C4901-100G）和0.1% BSA的消化液中，25℃消化4小时，酶解得到原生质体； 
(3)筛网过滤：将步骤(2)消化后的悬液置于碎冰上，然后先使用70μm(BD Falcon，352350) 筛网，再使用30μm(Miltenyi，130-098-458) 筛网过滤，100G，4℃，5min离心，弃消化液；
(4) 去杂洗涤：向步骤(3)得到的原生质体沉淀中加入1ml 4℃ W5溶液（154mM NaCl，125mM CaCl 2，5mM KCl，4mM MES，pH5.7），100G，4℃，5min离心，弃上清，得到的原生质体沉淀中加入1ml 4℃ W5溶液，100G，4℃，5min离心，弃上清，留50μl； 
(5) 细胞重悬：向步骤(4)离心后的细胞沉淀中加入1ml W5溶液进行重悬，镜检后用宽口的胶头滴管轻柔吹打混匀，通过稀释或浓缩将原生质体悬液调整至2×10 6 cells/ml。 
结果及分析：
细胞计数采用台盼蓝染色，0.4％的台盼蓝与细胞悬液按1:1混匀，用移液枪吸取 10μl加到血球计数板的加样孔中计数，显微镜观察计数区的细胞个数， 计算方式为((S1+S2+S3+S4+S5+S6+S7+S8+S9)/9/50*1000)个cells/μl。注：S1，S2，S3，S4，S5，S6，S7，S8，S9分别代表9个计数区的细胞个数。
在对比例2中，采用常规的解离方法制备的原生质体呈单个不同大小的圆形状态，活原生质体为透亮且轮廓清晰的圆，光镜下死原生质体被染上蓝色。视野中细胞均匀分散开，但有较多结团及细胞碎片，细胞活率较低（表2所示）。
                                        表2实施例2和对比例2实验效果对比
显然，本发明的上述实施例仅仅是为更清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定，对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其他不同形式的变化或变动，这里无法对所有的实施方法予以穷举，凡是属于本发明的技术方案所引申出的显而易见的变化或变动仍处于本发明的保护范围之列。

Claims (10)

  1. 一种拟南芥叶片样本组织解离液，所述组织解离液是在8%-10%甘露醇溶液中添加1％-3％纤维素酶、1％-3％果胶酶。
  2. 根据权利要求1所述的一种拟南芥叶片样本组织解离液，其特征在于，所述组织解离液是添加有2％纤维素酶、2％果胶酶的8%甘露醇溶液。 
  3. 一种拟南芥叶片样本的解离方法，包括：
    (1)表面清洗：将拟南芥叶片样本用甘露醇溶液进行表面清洗； 
    (2)样本剪碎：将步骤(1)清洗后的叶片置于无菌培养皿中，加入甘露醇溶液浸没过叶片、将叶片切成细条状； 
    (3)酶解消化：将步骤(2)得到的叶片细条转移至组织解离液中酶解得到原生质体，所述组织解离液是添加有1％-3％纤维素酶、1％-3％果胶酶的8%-10%甘露醇溶液； 
    (4)筛网过滤：将步骤(3)消化后的悬液置于碎冰上，然后筛网过滤、离心富集、弃消化液； 
    (5)去杂洗涤：向步骤(4)得到的原生质体沉淀中加入甘露醇溶液离心洗涤； 
    (6)细胞重悬：向步骤(5)离心后的细胞沉淀中加入甘露醇溶液进行重悬。
  4. 根据权利要求3所述的一种拟南芥叶片样本的解离方法，其特征在于，步骤(1)中，从拟南芥植株中取2-3cm长的叶片2-3片，用甘露醇溶液进行表面清洗2-3次，每次2-3min。
  5. 根据权利要求3所述的一种拟南芥叶片样本的解离方法，其特征在于，步骤(3)中，酶解温度为室温，酶解时间为4-5h。
  6. 根据权利要求3或5所述的一种拟南芥叶片样本的解离方法，其特征在于，步骤(3)中，酶解在转速10-15rpm的杂交炉中进行。
  7. 根据权利要求3所述的一种拟南芥叶片样本的解离方法，其特征在于，步骤(4)中，先使用70μm筛网过滤，再使用30μm筛网过滤。
  8. 根据权利要求3所述的一种拟南芥叶片样本的解离方法，其特征在于，步骤(4)和(5)中，离心条件均为：离心力100g，离心温度4-10℃，离心时间2-5min。
  9. 根据权利要求3所述的一种拟南芥叶片样本的解离方法，其特征在于，步骤(6)中，所述重悬的具体步骤为弃上清后留50μl，加入1ml 8%甘露醇溶液，用宽口的胶头滴管轻柔吹打混匀。
  10. 根据权利要求3所述的一种拟南芥叶片样本的解离方法，其特征在于，步骤(1) 、(2)、 (5)和(6)中，使用的甘露醇溶液的质量体积浓度均为8%。
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